Neuronal loss and brain atrophy in mice lacking B and L

Ute Felbor*†‡, Benedikt Kessler§, Walther Mothes¶, Hans H. Goebelʈ, Hidde L. Ploegh§, Roderick T. Bronson**, and Bjorn R. Olsen*

Departments of *Cell Biology and §Pathology, Harvard Medical School, Boston, MA 02115; †Institute of Human Genetics, University of Wu¨rzburg, D-97074 Wu¨rzburg, Germany; ¶Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536-0812; ʈDepartment of Neuropathology, University of Mainz, D-55131 Mainz, Germany; and **Tufts University School of Veterinary Medicine, North Grafton, MA 01536

Edited by Carla J. Schatz, Harvard Medical School, Boston, MA, and approved April 3, 2002 (received for review November 28, 2001) Cathepsins B and L are widely expressed cysteine impli- from those seen in classical Alzheimer’s disease and neuronal cated in both intracellular and extracellular matrix ceroid lipofuscinoses (NCLs). Our data demonstrate that cathe- remodeling. However, specific roles remain to be validated in vivo. psins B and L are required for integrity of the postnatal central Here we show that combined deficiency of cathepsins B and L in nervous system and clarify previous assumptions concerning the mice is lethal during the second to fourth week of life. biology of cathepsins B and L. The observed phenotype is, to our -B؊/؊͞L؊/؊ mice reveal a degree of brain atrophy not previously knowledge, without precedent in mice and provides an early seen in mice. This is because of massive apoptosis of select neurons onset mouse model of brain atrophy. in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive Materials and Methods astrocytosis and is preceded by an accumulation of ultrastructur- Genotyping. Mice examined in this study were from a mixed ally and biochemically unique lysosomal bodies in large cortical genetic background (C57BL͞6 ϫ 129͞sv). The experiments were neurons and by axonal enlargements. Our data demonstrate a performed in compliance with the regulations of the Harvard pivotal role for cathepsins B and L in maintenance of the central Medical School Standing Committee on Animals. The neo- nervous system. expression cassettes had been inserted into exon 4 of the and L upstream of the coding region for the athepsins B and L are widely expressed cysteine proteases. of the (see ref. 10; AstraZeneca, London). In vitro studies using purified enzymes and cysteine PCR analyses were performed with primers CTSLex3F (com- C Ј inhibitors suggested roles for these cathepsins in lysosomal mon oligonucleotide; 5 -CATGGAGATGAACGCCTTTGG- Ј Ј protein turnover at acidic pH. In a number of pathological 3 ), CTSLex4R (wild-type oligonucleotide; 5 -ATAGCCATTC- Ј conditions, both cathepsins are reported to be secreted and ACCACCTGCC-3 ), CTSL-PGK-neo (mutant oligonucleotide; Ј Ј involved in degradation of extracellular matrix components (1, 5 -ATCGCCTTCTTGACGAGTTCTTC-3 ), CTSBex4F (com- Ј Ј 2). More specific functions have recently been postulated in a mon oligonucleotide; 5 -GGTTGCGTTCGGTGAGG-3 ), Ј wide range of physiological and pathological processes. These CTSBex4R (wild-type oligonucleotide; 5 -AACAAGAGCCG- Ј include antigen presentation (3), prohormone processing (4), CAGGAGC-3 ), and CTSB-pMC1-neo (mutant oligonucleo- Ј Ј turnover of ␤-amyloid in Alzheimer’s disease (5), tumor invasion tide; 5 -CGATCCCATATTGGCTGC-3 ). (6), tumor angiogenesis (7), and apoptosis (8, 9). An in vivo validation of specific roles for cathepsins B and L Histology and Immunohistochemistry. Fifty double-mutant mice proved to be difficult, likely due to overlapping substrate spec- and their littermates were analyzed morphologically. Affected ϭ ificities of cysteine-type cathepsins. Cathepsin B null mice show animals at postnatal day 10.5 (P10.5) and older (n 19) were no obvious phenotype (10), but a reduction in premature generally killed when they were close to death as measured by intrapancreatic trypsinogen activation (11) and increased resis- weight loss of up to one-fourth of their body weight and reduced Ϫ/Ϫ͞ Ϫ/Ϫ tance to tumor necrosis factor-␣-mediated hepatocyte apoptosis activity. Of these, 11 cathepsin B L mice represented the (12) were observed under experimental conditions. Cathepsin period in which double-mutants died if litters were not reduced ϩ Ϫ/Ϫ͞ Ϫ/Ϫ L-deficient mice present only with a reduction in CD4 T cells (P10.5–17.5). Eight cathepsin B L mice between P21.5 (13) and recurrent hair loss (14). In contrast, and P50.5 were derived from breedings with reduced litters deficiency causes pyknodysostosis in mice and humans, consis- representing the stages in which brain shrinkage became visible. tent with its expression in and function in bone To determine the age of onset of each of the phenotypes, 31 resorption (15, 16). In addition, loss-of-function mutations in the double-mutant mice and their littermates between E17.5 and BIOCHEMISTRY human cathepsin C have been associated with Papillon– P9.5 were studied. For initial histological analyses, tissues were Lefe`vresyndrome, an autosomal recessive palmoplantar kera- fixed in Bouin’s fixative (Sigma). Multiple coronal brain sections Ϫ/Ϫ͞ Ϫ/Ϫ tosis with periodontopathia (17). were taken of each animal. Several cathepsin B L brains To assess whether cathepsins B and L compensate for each were serial sectioned. Samples of all tissues were analyzed from Ϫ/Ϫ͞ Ϫ/Ϫ other and to identify specific roles for these two proteases in vivo, six cathepsin B L animals (P12.5 twice, P17.5, P24.5, and all organs of mice lacking from one to four cathepsin B and L P50.5 twice). In addition, brains of 5-, 8-, and 12-month-old Ϫ/Ϫ͞ ϩ/Ϫ ϩ/Ϫ͞ Ϫ/Ϫ Ϫ/Ϫ͞ ϩ/ϩ ϩ/ϩ͞ Ϫ/Ϫ alleles were analyzed at various ages. Despite ubiquitous expres- cathepsin B L ,B L ,B L ,B L , and ϩ/ϩ͞ ϩ/ϩ sion of the two proteases, homozygous double-mutant mice B L mice were examined. Paraffin-embedded sections exhibit a selective neuronal vulnerability with rapid loss of were stained with hematoxylin and eosin (H&E), the periodic cerebral cortical neurons as well as cerebellar Purkinje and granule cells. Dying cells display nuclear condensation, DNA This paper was submitted directly (Track II) to the PNAS office. fragmentation, and other features characteristic of apoptosis. As Abbreviations: GFAP, glial fibrillary acidic protein; H&E, hematoxylin͞eosin; LFB, luxol fast observed in human neurodegeneration and aging, fading of blue; NCL, neuronal ceroid lipofuscinosis; Pn, postnatal day n; TUNEL, terminal deoxynu- functional neurons is accompanied by an increase of glial tissue cleotidyl -mediated dUTP nick-end labeling. and preceded by the occurrence of intraneuronal inclusions. ‡To whom reprint requests should be addressed. E-mail: [email protected] These are, however, ultrastructurally and biochemically distinct wuerzburg.de.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.112632299 PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 7883–7888 Downloaded by guest on September 30, 2021 acid͞Schiff reagent method counterstained with hematoxylin, and luxol fast blue in combination with cresylecht violet. Glial fibrillary acidic protein (GFAP)-immunohistochemistry was performed on P17.5, P21.5, P24.5, and P50.5 paraffin sections with polyclonal rabbit anti-cow GFAP (1:200, Dako) using the Vectastain Elite ABC kit and the diaminobenzidene substrate kit from Vector Laboratories. The antibody against subunit c of the mitochondrial F1F0-ATP synthase was kindly provided by E. Kominami (Department of Biochemistry, Juntendo University, Tokyo, Japan) and used as described on P17.5 brain sections (18).

In Situ Labeling of Fragmented DNA. P16.5, P17.5, P23.5, and P24.5 double-mutant and control brains were frozen in OCT embed- ding medium (Tissue-Tek, Sakura Finetek Europe, B.V., Zoeter- woude, The Netherlands), serial sectioned, and thaw-mounted on Polysine slides (Menzel Gla¨ser, Braunschweig, Germany). Apoptotic cells were identified with the terminal deoxynucleo- tidyltransferase-mediated dUTP nick-end labeling (TUNEL) method by using the ApopTag Plus peroxidase In Situ apoptosis detection kit from Intergen (Oxford, U.K.) on 8-␮m frozen sections representative of all major brain regions.

Electron Microscopy. Brains of P7.5, P12.5, and P24.5 cathepsin BϪ/Ϫ͞LϪ/Ϫ mice were fixed by intracardiac perfusion of Kar- novsky’s fixative. One cerebral hemisphere was processed for paraffin embedding and histopathological analysis. Semithin Epon-embedded sections of the other hemisphere were stained with toluidine blue for orientation. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a 1200EX JEOL electron microscope.

Biochemical Analyses. Brains and spleens from P10.5, P12.5, P13.5, P17.5, and P18.5 representative mice of all nine genotypes were snap frozen and kept at Ϫ80°C. Tissues were homogenized in ice-cold PBS (10 ml͞g of tissue) with five passes of a Teflon͞glass Dounce homogenizer. The tissue homogenates were washed twice with PBS and pellets were kept at Ϫ80°C. Preparation of the 125I-radiolabeled active-site-directed probes LHVS-PhOH and JPM-565, lysis of brain tissue homog- enates, and labeling with the radioactive probes were performed as reported (19). Palmitoyl-protein-thioesterase- and tripeptidyl- peptidase-I activities were determined by using the fluorogenic substrates MU-6S-palm-␤Glc (4-methylumbel- liferyl-6-thiopalmitoyl ␤-D-galactopyranoside) (Moscerdam, Rotterdam, The Netherlands) and AAF-AMC (Ala-Ala-Phe-7- amido-4-methylcoumarin) (Sigma), respectively, and the assays were performed twice in triplicate as described (20–22). Fig. 1. Early death in cathepsin BϪ/Ϫ͞LϪ/Ϫ mice. (a) Genotyping of wild-type Subcellular fractionations, preparations of enriched fractions (lanes 1), cathepsin BϪ/Ϫ͞LϪ/Ϫ (lanes 2), and cathepsin Bϩ/Ϫ͞Lϩ/Ϫ (lanes 3) mice of dense bodies, and preparations of proteolipids were per- by PCR. (b) 125I-LHVS-PhOH and 125I-JPM565 active-site labeling of brain ho- formed as described (23–25). The anti-cathepsin D antibody mogenates from cathepsin Bϩ/Ϫ͞Lϩ/ϩ (lane 1), cathepsin Bϩ/Ϫ͞Lϩ/Ϫ (lane 2), ϩ Ϫ Ϫ Ϫ ϩ ϩ ϩ Ϫ ϩ Ϫ ϩ Ϫ used for Western blot analysis was supplied by Santa Cruz cathepsin B / ͞L / (lane 3), cathepsin B / ͞L / (lane 4), cathepsin B / ͞L / Ϫ/Ϫ͞ ϩ/Ϫ Biotechnology. (lane 5), and cathepsin B L (lane 6) mice. Cathepsins B and L (CTSB and CTSL) and their corresponding proforms were identified according to previous Results studies (19, 24). (c) Mortality diagram of wild-type (n ϭ 20, dashed line) and cathepsin BϪ/Ϫ͞LϪ/Ϫ mice (n ϭ 14, solid line). Generally, the lifetime could be ؊/؊͞ ؊/؊ Cathepsin B L Mice Die in Infancy. Mice homozygous for extended by only a few days through reduction of litter size to 3 to 5 pups at cathepsin B and L deficiency were generated by intercrossing day 7 in further breedings (II) (n ϭ 34). Importantly however, these breedings ϩ Ϫ ϩ Ϫ cathepsin B / ͞L / mice and genotyped by PCR (Fig. 1a). yielded 8 mice aged 21 to 50 days in which brain shrinkage became evident. Labeling of tissue homogenates with the thiol protease-specific active site probes 125I-LHVS-PhOH and 125I-JPM-565 confirmed that these proteins were absent in null mice (Fig. 1b). The emaciated state (Fig. 1c). No animal lacking all four cathepsin numbers of offspring of each of the nine possible genotypes were B and L alleles was found among 307 mice genotyped at day 21, in accord with Mendelian segregation at birth (5 double-mutants whereas the expected proportions of all other genotypes were of 77 genotyped newborn animals). Newborn mice of all geno- obtained. types were indistinguishable visibly. However, cathepsin BϪ/Ϫ͞ The lifespan of double-mutant mice increased by several days LϪ/Ϫ mice gained weight only slowly after day 5, and their body when healthy littermates were removed at day 7 (Fig. 1c). At this weight started to decline during the second week of life. Most stage, growth impairment, combined with a delay in fur devel- homozygous double-mutant mice died around day 12 in an opment, enabled the identification of the cathepsin BϪ/Ϫ͞LϪ/Ϫ

7884 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112632299 Felbor et al. Downloaded by guest on September 30, 2021 Fig. 2. Cerebral and cerebellar atrophy in cathepsin BϪ/Ϫ͞LϪ/Ϫ mice. (a–c) Luxol fast blue (LFB)-stained coronal sections of P50.5 littermate control (a), P17.5 (b), and P50.5 (c) cathepsin BϪ/Ϫ͞LϪ/Ϫ cerebrum demonstrating cortical shrinkage accompanied by white-matter (wm) reduction between days 17 and 50 in double-mutants (cx ϭ cortex; scale bar in a ϭ 1 mm). The deep cerebral white matter is not yet myelinated in 17-day mutants (b). (d–f) LFB-stain of P50.5 wild-type (d), P17.5 (e), and P50.5 (f) cathepsin BϪ/Ϫ͞LϪ/Ϫ cerebellum (m ϭ molecular layer, g ϭ granular layer; scale bar ϭ 1 mm). (h–j) H&E stain of the above samples at ϫ100. Note the dying bright red Purkinje cell in i, the complete absence of the Purkinje cell layer (p), and the striking reduction in cellular density in the granule cell layer (g) in j.(g) Nuclear pyknosis, karyorrhexis, and brightly red-stained angular cytoplasm in dying cortical neurons at P24.5. Healthy neurons exhibit a lightly stained nucleus and a darkly stained nucleolus as seen in the bottom part of g.(k) Lipid-laden macrophages in the corpus callosum at P50.5 (ϫ1250 in g–k).

phenotype. Four cathepsin BϪ/Ϫ͞LϪ/Ϫ mice survived the wean- death, TUNEL (26) was performed. Widespread TUNEL- ing period but depended on continuous nursing with wetted food positive staining was found in the serial sectioned P23.5–24.5 twice daily. These mice demonstrated hesitating voluntary cathepsin BϪ/Ϫ͞LϪ/Ϫ cerebral cortex and the cerebellar granule- movements, a mild unusual tremor, and subtle rear limb spas- cell layer (Fig. 3 b, c, and e), with only an occasional TUNEL- ticity. Characteristically, they swayed backwards while grooming positive cell in the cerebellar molecular cell layer (data not and used their tail for balance. Animals of both sexes were killed shown). This staining was absent from wild-type control sections at day 50, weighing less than half of the expected body weight. (Fig. 3 a and d). While intact up to postnatal day 16, the Thus, reduction of competitors in the cage and special animal Purkinje-cell monolayer had largely vanished in P21.5–24.5 care enabled us to study the pathological changes from late double-mutant mice. However, P17.5 cathepsin BϪ/Ϫ͞LϪ/Ϫ brain embryonal stages (E17.5͞E18.5) through P50.5. sections revealed some TUNEL-positive cells in the Purkinje- cell monolayer (data not shown). At this stage, significantly -Cerebral and Cerebellar Atrophy in Cathepsin B؊/؊͞L؊/؊ Mice. Be- fewer TUNEL-positive granule cells were observed when com sides hyperproliferation of keratinocytes as described for ca- pared with P23.5–24.5 double-mutant sections. This observation thepsin L-deficient mice (14) and an unspecific involution of the suggests that Purkinje cells are the more likely first target. thymus in terminally ill animals, histology of all organs revealed Neuronal loss was paralleled by an increasing occurrence of Ϫ Ϫ Ϫ Ϫ pathological features only in the brains of cathepsin B / ͞L / hypertrophic astrocytes and Bergmann glia as demonstrated by mice. immunostaining with an antibody against GFAP. Fifty-day-old BIOCHEMISTRY Fifty-day-old mice exhibited a pronounced cerebral and cer- double-mutant mice showed the most striking reactive gliosis in ebellar atrophy. The total thickness of the cerebral cortex was the cerebral cortex (Fig. 3g). Consistent with the almost com- significantly reduced when compared with that of wild-type or plete loss of Purkinje cells, the P24.5 and P50.5 cathepsin younger double-mutant mice (compare Fig. 2c with Fig. 2 a and BϪ/Ϫ͞LϪ/Ϫ cerebellar cortex showed the picture of an isomorphic b). On cut surfaces, the cerebral white matter could hardly be gliosis (Fig. 3i). distinguished from gray matter and proved to be thinned in Luxol fast blue (LFB)-stained sections (Fig. 2c). Furthermore, Accumulation of Membranous Compartments in Large Neurons and the cerebellar molecular and internal granule cell layers were Axons. Before TUNEL reactivity and cell death, cell bodies of strikingly reduced (compare Fig. 2f with Fig. 2 d and e), and the about 50% of neurons in the cerebral cortex accumulated Purkinje-cell layer had disappeared (compare Fig. 2j with Fig. eosinophilic, periodic acid Schiff-, toluidine blue-, and LFB- 2h). These changes were the result of massive neuronal death in positive material. The amount of inclusion material increased the cerebellar Purkinje- and granule-cell layers (Fig. 2i) and the rapidly between P11.5 and P15.5 (Fig. 4 a and b). Already at P7.5, cerebral cortex (Fig. 2g) during the third and fourth week of life. a large subset of Purkinje cells showed a haze of LFB stain and To assess whether the numerous pyknotic and karyorrhectic some granules (data not shown). Apart from occasional inclu- nuclei seen in H&E stained neurons (Fig. 2 g and i) have sions in the CA3 region, the hippocampus was spared. Similarly, fragmented DNA indicative of an apoptotic mechanism of cell only a few larger neurons in the thalamus and basal ganglia and

Felbor et al. PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 7885 Downloaded by guest on September 30, 2021 ing as round globules of less than 20 ␮m in axon-rich areas such as the corpus callosum, fornix, rostral commissure, fimbriae hippocampi, and deep cerebellar white matter (Fig. 4 f–h). Electron microscopy of the 12-day-old cathepsin BϪ/Ϫ͞LϪ/Ϫ corpus callosum revealed expanded axons that contained a large number of dense bodies often replete with lamellae and mito- chondria. Preexisting microtubules were displaced to the periph- ery of the cell process (Fig. 4i). While unique in location and dense packing, the profiles resembled axonal inclusions seen in other lysosomal diseases (27, 28). Cathepsin BϪ/Ϫ͞LϪ/Ϫ mice also showed a hippocampal lam- ination defect. Whereas the Ammon’s horn of the hippocampus usually presents as a morphologically distinct C-shaped struc- ture, the CA3 region of the stratum pyramidale was broadened and split in double-mutant mice older than P3.5 (U.F., R.T.B, and B.R.O, unpublished data). Variable phenotypes in cathepsin BϪ/Ϫ͞LϪ/Ϫ mice included white matter hypodensities, a hydro- cephalus ex vacuo, and a cavity forming between the corpus callosum and the ventral commissure of the fornix and extending rostrally to divide the septal nuclei (data not shown). The brains of 5-, 8-, and 12-month-old cathepsin BϪ/Ϫ͞Lϩ/Ϫ,Bϩ/Ϫ͞LϪ/Ϫ, BϪ/Ϫ͞Lϩ/ϩ,Bϩ/ϩ͞LϪ/Ϫ, and Bϩ/ϩ͞Lϩ/ϩ mice showed neither neuronal loss, nor intraneuronal LFB-positive inclusions, nor a hippocampal split, indicating that the observed phenotype re- quires the absence of both cathepsin B and L activity.

.The Cathepsin B؊/؊͞L؊/؊ Phenotype Is Distinct from Classical NCLs The early and severe brain atrophy is highly reminiscent of NCLs, the most common group of autosomal-recessively inher- ited neurodegenerative disorders in children and young adults (29). However, lipopigment accumulation in NCLs is found in retina, skin, myocardium, and visceral organs which were incon- spicuous in LFB-stained cathepsin BϪ/Ϫ͞LϪ/Ϫ sections up to day 50. In addition, the ultrastructural morphology of the accumu- Ϫ/Ϫ͞ Ϫ/Ϫ Fig. 3. Positive TUNEL-labeling indicative of apoptotic cell death and posi- lated material in cathepsin B L neurons did not unequiv- tive GFAP immunostaining demonstrating reactive gliosis in response to neu- ocally resemble the granular, curvilinear, or fingerprint profiles ron death in cathepsin BϪ/Ϫ͞LϪ/Ϫ mice. (a–c) Control (a) and cathepsin BϪ/Ϫ͞ seen in NCLs (30). Furthermore, the activities of palmitoyl LϪ/Ϫ P24.5 (b and c) cryosections of the cerebral cortex showing small TUNEL- protein thioesterase (31) and I (32), the positive globules in the center of a shrunken cathepsin BϪ/Ϫ͞LϪ/Ϫ neuron in b only two enzymes known to be mutated in NCLs, were normal and division into apoptotic cell fragments in c.(d and e) Control (d) and in 12-day-old and elevated in older cathepsin BϪ/Ϫ͞LϪ/Ϫ brain Ϫ Ϫ Ϫ Ϫ cathepsin B / ͞L / (e) P24.5 granule layer of the cerebellar central lobe lysates (see Fig. 6, which is published as supporting information shows TUNEL-positive material around the periphery of double-mutant nu- on the PNAS web site, www.pnas.org). Immunohistochemistry clei, suggesting chromatin margination. (f–i) GFAP-immunostaining of 6-␮m coronal paraffin sections reveals abundant star-shaped hypertrophic astro- with an antibody against subunit c of the mitochondrial F1F0- cytes in the 50-day-old double-mutant cerebral cortex (g) not present in the ATP synthase (18), a standard marker for NCLs, showed no control cortex (f). Control (h) and double-mutant (i) 50-day-old cerebellum relevant staining in the cerebral and cerebellar cortex of cathep- Ϫ/Ϫ Ϫ/Ϫ shows the picture of an isomorphic gliosis in the latter. The processes of the sin B ͞L mice (data not shown). Accordingly, dense-body Ϫ Ϫ ϩ ϩ ϩ ϩ Bergmann glia are in parallel orientation. Note the tiny healthy area in i with preparations (25) from wild-type, cathepsin B / ͞L / ,B / ͞ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ a residual Purkinje cell (arrow). [Scale bars ϭ 7 ␮m(a–e), and 0.1 mm (f–i).] L / , and B / ͞L / mice separated on SDS and Tris-Tricine polyacrylamide gels, proteolipid fractions prepared from dense bodies (25), and subcellular fractions of total brain homogenates select nuclei in the brainstem revealed some granules. The spinal (23) did not show any specific accumulation of polypeptides such cord and peripheral ganglia were largely unaffected. There was as the subunit c of the mitochondrial F1F0-ATP synthase (data no motoneuron loss or neurogenic muscular atrophy, consistent not shown). with the observation that cathepsin BϪ/Ϫ͞LϪ/Ϫ mice do not experience flaccid paralysis. Procathepsin D and Cathepsin D Are Increased in Cathepsin B؊/؊͞L؊/؊ Ultrastructurally, intraneuronal inclusions of 12-day-old Brains. Ϫ Ϫ Ϫ Ϫ Accumulation of ceroid-lipofuscinous material was ob- cathepsin B / ͞L / mice consisted of electron-dense, often served in sheep with an enzymatically inactive form of the membrane-bound bodies of varying size and shape occasionally cathepsin D (33) and in mice with a truncated containing lamellae (Fig. 4 c and d), suggesting lysosomal cathepsin D polypeptide (34, 35). The accumulating inclusions in residual bodies. These inclusions formed circumscribed, but cathepsin BϪ/Ϫ͞LϪ/Ϫ mice have a significantly earlier onset and loosely arranged, groups within the neuronal perikarya. Later, a much more rapid progression resulting in apoptotic neuronal they occupied almost the entire perikaryon, displacing and death and brain shrinkage not reported for cathepsin D-deficient replacing other organelles such as mitochondria (P24.5, Fig. 4e). mice. Because in vitro studies suggested, however, that the The remaining neurons in 50-day-old mice were largely devoid conversion of a 47-kDa cathepsin D intermediate into the of LFB-positive inclusions. mature 31-kDa enzyme might be accelerated by cysteine pro- A hallmark of double-mutant mice, observed as early as day teases after its transfer into dense lysosomes (36), we assessed 3 and preceding all aforementioned changes, was a dense whether such processing is impaired in brains lacking cathepsins accumulation of eosinophilic and LFB-positive material appear- B and L. Western-blot analyses showed that processing of

7886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112632299 Felbor et al. Downloaded by guest on September 30, 2021 Fig. 5. Endosomal and lysosomal compartments accumulate proportionally in null mice because processing of the short-lived 53-kDa precursor of cathep- sin D into the 47-kDa intermediate form and the mature enzyme composed of a 31-kDa and a 14-kDa fragment is not altered in cathepsin BϪ/Ϫ͞LϪ/Ϫ brain lysates. Ten micrograms of brain homogenates from wild-type (WT), 10-, and 18-day-old cathepsin BϪ/Ϫ͞LϪ/Ϫ mice (MUT) were separated on 7.5–17.5% SDS͞PAGE gels, electroblotted onto nitrocellulose, and probed with an anti- cathepsin D antibody.

procathepsin D is not blocked in cathepsin BϪ/Ϫ͞LϪ/Ϫ brains (Fig. 5). Since the intermediate and mature forms of cathepsin D serve as organelle markers for prelysosomal and lysosomal compartments, respectively, the observed proportional increase of both forms in cathepsin BϪ/Ϫ͞LϪ/Ϫ brains suggests that endosomal and lysosomal compartments accumulate with time. Discussion This study demonstrates that cathepsins B and L are essential for maturation and integrity of the postnatal central nervous system (CNS) and that the two proteases compensate for each other in vivo. Consistent with an increase of cathepsin B and L mRNA expression from normal newborn to 5-day-old mouse brains (37) and cathepsin B and L mRNA coexpression in rat brain neurons (38), cathepsin BϪ/Ϫ͞LϪ/Ϫ mice exhibit histopathological alter- ations in the CNS that change rapidly as a function of time. The dramatic course of neurodegeneration in infant cathepsin BϪ/Ϫ͞ LϪ/Ϫ knockouts is unlike neuronal loss in, e.g., Munc18–1-deficient mice, which show a failure in prenatal development (39). It is also distinct from known mouse models of human NCLs, which have late disease onset and do not reveal cytological changes indicative of neuronal cell death and brain atrophy (40). BIOCHEMISTRY Cysteine proteases whose active-site sulfhydryl groups are oxidation-sensitive have been implicated in NCL etiology as well as in decreased protein turnover in aging brains. Intraventricular infusions of the broad-spectrum serine and

expanded and filled with LFB-positive inclusions. (c–e) Electron micrographs of inclusions in P12.5 (c and d) and P24.5 (e) cathepsin BϪ/Ϫ͞LϪ/Ϫ cortical neurons. (d) Higher magnification of the inclusion indicated by an arrow in c.(f) Sagittal section of the P11.5 double-knockout brain shown in a revealing a dense accumulation of eosinophilic globules in cerebral white matter (H&E stain). (g and h) Magnification of the area surrounding the asterisk shown in f stained Fig. 4. Accumulation of amorphous and membranous material in neuronal with H&E and LFB, respectively. Normal white matter is seen in the upper right perikarya, dendrites, and axons in cathepsin BϪ/Ϫ͞LϪ/Ϫ mice. (a and b) LFB stain corner. All LFB stainings were performed simultaneously. (i) Electron micro- of P11.5 and P15.5 double-mutant cortical neurons. At P11.5 (a), only a few graph of membranous inclusions in a swollen axon in the corpus callosum of inclusions are observed in some neuronal perikarya (arrow). The arrowhead a P12.5 cathepsin BϪ/Ϫ͞LϪ/Ϫ mouse. [Scale bars ϭ 15 ␮m(a, b, g, h), 2 ␮m(c), points to a normal neuron. Only a few days later, at P15.5 (b), the perikarya are 100 nm (d), 1.6 ␮m(e), 0.15 mm (f), and 500 nm (i).]

Felbor et al. PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 7887 Downloaded by guest on September 30, 2021 inhibitor leupeptin and the cysteine protease inhibitor E-64c protective response to earlier pathogenetic mechanisms. Taking resulted in a general accumulation of dense granular aggregates into account that the pH range of cathepsins B and L should throughout rodent brains, as well as in the retina and internal allow activity in less acidic endosomal compartments (1, 2), it is organs, thereby partially reproducing features of NCLs, Alzhei- conceivable that these two cathepsins are more widely involved mer’s disease, and aging (41). Several in vitro studies using in the homeostasis of vesicle trafficking and formation of axons cultured hippocampal and cortical slices (42, 43) or cardiac and synaptic connections during early postnatal life. The early myocytes (44) appeared to reproduce the in vivo experiments. In onset and rapid progression of neurodegeneration should make Ϫ Ϫ Ϫ Ϫ contrast, cathepsin BϪ/Ϫ͞LϪ/Ϫ mice show no general pleiotropic cathepsin B / ͞L / mice ideal for further studies into the effects in membrane turnover within their lifetime. One possible neurobiology of these enzymes and for evaluation of therapeutic explanation for the selective neuronal vulnerability in cathepsin strategies designed to counteract apoptosis or to replace dying or BϪ/Ϫ͞LϪ/Ϫ knockouts is that the inhibitors also affect additional lost neurons. enzymes. Ϫ/Ϫ͞ We thank C. Peters and J. Deussing for providing cathepsin BϪ/Ϫ mice, An initial biochemical characterization of cathepsin B Ϫ Ϫ LϪ/Ϫ mice revealed no obvious metabolic substrate accumulating AstraZeneca for / mice, E. Kominami for the antibody in cathepsin BϪ/Ϫ͞LϪ/Ϫ brains. It remains to be clarified whether against subunit c of the mitochondrial F1F0-ATP synthase, and N. the earliest and pathognomonic finding, the accumulation of Ru¨ttling, L. Zhang, and M. Ericsson for expert assistance with the Ϫ/Ϫ͞ Ϫ/Ϫ histotechnology and electron microscopy. This work was supported by membranous compartments in cathepsin B L axons Deutsche Forschungsgemeinschaft Grant Fe 432͞6-1 (to U.F.), the throughout the corpus callosum far distant from neuronal Human Science Frontier Program Organisation (B.K.), Hoffman La perikarya, as well as the perikaryal inclusions in large neurons, Roche (B.K.), Entremed, Inc. (B.R.O.), and the National Institutes of is causally related to neurodegeneration or result from the cell’s Health (H.L.P. and B.R.O.).

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